The artificial organization of inorganic material into designed structures on the nanometer scale is a fundamental technological problem. Photolithography, the predominant technique, is used primarily to achieve high-density information storage and processing. Further mastery of architectures on this length scale may advance these goals and create new realms of application, such as a general approach to interfaces with single biomolecules or organelles.
Sequence-specific polymers are a promising pathway toward the goal of artificial organization of inorganic material, as demonstrated by the DNA origami technique, in which a long single strand of DNA is folded into elaborate patterns using short oligomers that connect different parts of the long strand. These structures have been formed into tiles and tubes in various forms demonstrating the versatility and generality of the method. Others have used the assembly properties of DNA to precisely organize inorganic nanoparticles in periodic or chiral structures (Nykypanchuk, D.; Maye, M. M.; van der Lelie, D.; Gang, O. “DNA-guided crystallization of colloidal nanoparticles,” Nature, 2008, v. 451, 549-552; Mastroianni, A. J.; Claridge, S. A.; Alivisatos, A. P. “Pyramidal and Chiral Groupings of Gold Nanocrystals Assembled Using DNA Scaffolds,” Journal of the American Chemical Society, 2009, v. 131 (24): 8455-8459). Gold nanoparticles have been incorporated into DNA origami and other nucleic acid nanostructures with high precision, but not with high generality, requiring constraints on the templating structure, the use of specific particle sizes, or requiring coatings that are thick with respect to inorganic particle size (Ding, B. Q.; Deng, Z. T.; Yan, H.; Cabrini, S.; Zuckermann, R. N.; Bokor, J. “Gold Nanoparticle Self-Similar Chain Structure Organized by DNA Origami,” Journal of the American Chemical Society, 2010, v. 132 (10): 3248-3249; Sharma, J.; Chhabra, R.; Cheng, A.; Brownell, J.; Liu, Y.; Yan, H. “Control of Self-Assembly of DNA Tubules Through Integration of Gold Nanoparticles,” Science, 2009, v. 323 (5910): 112-116; Zheng, J. W.; Lukeman, P. S.; Sherman, W. B.; Micheel, C.; Alivisatos, A. P.; Constantinou, P. E.; Seeman, N. C. “Metallic Nanoparticles Used to Estimate the Structural Integrity of DNA Motifs,” Biophysical Journal, 2008, v. 95 (7): 3340-3348; Wen, Y. Q.; McLaughlin, C. K.; Lo, P. K.; Yang, H.; Sleiman, H. F. “Stable Gold Nanoparticle Conjugation to Internal DNA Positions: Facile Generation of Discrete Gold Nanoparticle—DNA Assemblies,” Bioconjugate Chemistry, 2010, v. 21 (8): 1413-1416).
One factor likely to limit the generality of assembly of inorganic materials using DNA, or their subsequent application in biological environments, is the need for using a divalent salt in concentration levels of at least tens of millimolar (mM) during assembly and manipulation of compact DNA nanostructures. This is especially true in the case of DNA origami, where tightly folded strands create high densities of negatively charged phosphate ions. In contrast, inorganic nanoparticles typically benefit from the effect of inter-particle electrostatic repulsion for stability against particle aggregation. However, increased salt concentration weakens this repulsive effect and divalent salts can also have a crosslinking effect as noted by Elimelech, et al., (Particle Deposition & Aggregation: Measurement, Modeling and Simulation; Butterworth-Heinemann: Oxford, 1998). Thick surfactant or polymer coatings on particles can mitigate this effect but use of such coatings may also mask the function of the inorganic material, limit the precision and pitch of positions on a template, and limit transport and uptake in biological environments.
For functionalizing gold nanoparticles with DNA, a small anionic ligand, bis (p-sulfonato) triphenylphosphine, is often used because it is a small molecule that is easily displaced by thiolated functional molecules and one that has low nonspecific binding to DNA and other materials. However, bis (p-sulfonato) triphenylphosphine provides no tolerance to even relatively low concentrations (e.g. ˜1 mM) of magnesium solute cation.
Another strategy is to completely encapsulate nanoparticles with thiolated DNA, in some cases applying a small number of long strands along with a large number of shorter oligomers (op. cit., D. Nykypanchuk; B. Q. Ding; and J. Sharma). This modification confers some magnesium ion tolerance together with some loss of the advantages of the phosphine. However, they also perturb DNA nanostructures: their structure in the presence of particles is very different from their structure in the absence of these particles (op. cit. Yan, H., et al. Science, 2009, v. 323 (5910): 112-116). This is not surprising given that the total charge on the functionalized gold particle is high, and not widely tunable.
Greater versatility may be obtained by using other sequence-specific polymers, with a wider range of functionality, as ligands. Short oligo-peptides have been designed by Fernig et al. that confer monovalent salt tolerance in the high hundreds of mM, although any tests of these in the presence of magnesium salts and/or DNA have not been reported (see Levy, R.; Thanh, N. T. K.; Doty, R. C.; Hussain, I.; Nichols, R. J.; Schiffrin, D. J.; Brust, M.; Fernig, D. G. “Rational and Combinatorial Design of Peptide Capping Ligands for Gold Nanoparticles,” Journal of the American Chemical Society, 2004, v. 126 (32): 10076-10084; Doty, R. C.; Tshikhudo, T. R.; Brust, M.; Fernig, D. G., “Extremely Stable Water-Soluble Ag Nanoparticles,” Chemistry of Materials, 2005, v. 17 (18): 4630-4635; and Duchesne, L.; Gentili, D.; Comes-Franchini, M.; Fernig, D. G. “Robust Ligand Shells for Biological Applications of Gold Nanoparticles,” Langmuir, 2008, v. 24 (23): 13572-13580). Oligo-N-functional glycines (also known as “peptoids”) have conformations that are less salt-dependent than similar peptides, and it was posited that this may confer function that is also less salt-dependent.
U.S. Pat. Nos. 6,306,993 and 6,759,387 disclose methods and compositions for transporting drugs and macromolecules across biological membranes. The invention includes a method for enhancing transport of a compound across a biological membrane, wherein a biological membrane is contacted with a conjugate containing a biologically active agent that is covalently attached to a transport polymer consisting of 6 to 25 subunits, at least 50% of which contain a guanidino- or an amidino-sidechain moiety. The latter also discloses polymers that include, for example, poly-arginine molecules that are preferably between about 6 and 25 residues in length.
U.S. Pat. No. 7,427,600 provides covalent attachment of active agents to a peptide. The invention may be distinguished from the above mentioned technologies by virtue of covalently attaching the active agent directly, which includes, for example, pharmaceutical drugs and nutrients, to the N-terminus, the C-terminus or to the side chain of an amino acid, an oligopeptide or a polypeptide, also referred to herein as a carrier peptide. In another embodiment, when the active agent is itself an amino acid active agent, then the active agent may be part of the chain at either the C-terminus or N-terminus through a peptide bond, or interspersed in the polypeptide via peptide bonds on both sides of the active agent. In another embodiment, the peptide stabilizes the active agent, primarily in the stomach, through conformational protection. In this application, delivery of the active agent is controlled, in part, by the kinetics of unfolding of the carrier peptide. Upon entry into the upper intestinal tract, indigenous enzymes release the active ingredient for absorption by the body by hydrolyzing the peptide bonds of the carrier peptide. This enzymatic action introduces the second phase of the sustained release mechanism.
Published U.S. Pat. Appln. No. 20070098713 discloses the use of small particles in biological systems, including the delivery of biologically active agents. Some embodiments relate to a collection of particles having an agent, a surfactant molecule having an HLB value of less than about 6.0 units, and a polymer, wherein the collection of particles has an average diameter of less than about 100 nanometers, wherein the agent is a protein, carbohydrate, polypeptide, adjuvant, nucleic acid encoding a protein, a visualization agent, or a marker.
Similarly, published U.S. Pat. Appln. No. 20080213377 discloses systems, methods, and compositions for targeted delivery of nanoparticles and/or agents to tissues, cells, and/or subcellular locales. In general, compositions comprise a nanoparticle (e.g. quantum dot, polymeric particle, etc.), at least one modulating entity (such as a targeting moiety, transfection reagent, protective entity, etc.), and at least one agent to be delivered (e.g. therapeutic, prophylactic, and/or diagnostic agent). The present invention provides methods of making and using nanoparticle entities in accordance with the present invention.
In addition, U.S. Pat. Appln. No. 20090226528 discloses methods and compositions for delivering payload molecules, including nucleic acids, using yeast cell wall particles. Embodiments of the invention are useful for delivering a variety of molecules to cells. Aspects of the invention include yeast cell wall particles encapsulating nanoparticles comprising payload molecules.